Pseudomonas aeruginosa is an opportunistic pathogen commonly isolated from patients with cystic fibrosis, nosocomial pneumonias, urinary tract infections, or severe burns. A primary determinant of P. aeruginosa virulence is a type III secretion system (T3SS). The T3SS is required for full virulence in animal infection models and functions by translocating toxins with anti-phagocytic and cytotoxic activities into host cells. The central regulator of T3SS gene expression is ExsA, a member of the AraC/XylS family of transcriptional activators. Although AraC/XylS family members regulate T3SS gene expression in a number of important pathogens, the mechanism of transcriptional activation by these family members is poorly understood. One reason for our limited knowledge is the tendency of AraC/XylS family members to become insoluble upon purification. Fortunately, we find that ExsA remains soluble and active following purification. This places us in a unique position to address the mechanism of transcriptional activation by ExsA. Increased understanding of transcriptional activation by ExsA, and of regulatory events that control ExsA activity, will shed light on the mechanism by which other signaling networks control T3SS gene expression in P. aeruginosa. The long-term goals of the work are to identify, characterize, and integrate regulatory systems involved in controlling P. aeruginosa T3SS gene transcription, and, armed with that knowledge, develop inhibitors of T3SS gene expression that can be used clinically to treat P. aeruginosa infections. Towards those goals we propose the three specific aims: (1) Characterize promoter elements required for ExsA binding and transcriptional activation using band shift and transcriptional reporters assays. These experiments will aid in the identification and characterization of promoter elements required for ExsA-binding and/or transcriptional activation. (2) Determine the mechanism of ExsA- dependent transcriptional activation. Fundamental questions regarding the mechanism of ExsA-dependent transcriptional activation will be addressed using footprinting and in vitro transcription assays. These questions include whether ExsA functions to recruit RNA polymerase to T3SS promoters and/or facilitates formation of an open transcriptional complex. (3) Define mechanisms involved in regulation of ExsA activity. ExsD and PtrA both directly bind ExsA to inhibit transcription. We will determine whether they function by preventing interacting with T3SS promoters or interfering with the activation properties of ExsA. Many medically important bacteria use a so-called type III secretion system to inject toxins into cells found in the human body during infections. There is considerable interest in developing therapeutic strategies that interfere with the ability of the bacteria to deploy the type III secretion system. This work will address the potential strategy of specifically preventing expression of genes required for proper functioning of the type III system.